News from the American Heart Association Meeting (AHA)

The ACC and AHA Launch National Alliance To Reduce Door-to-Balloon (D2B) Times

The American College of Cardiology (ACC), together with the American Heart Association (AHA) and other key national healthcare organizations, announced the launch of its latest quality campaign, Door to Balloon (D2B): An Alliance for Quality. A Guidelines Applied in Practice (GAP) Program, the D2B campaign is aimed at improving the timeliness of lifesaving therapy for patients with heart attacks at U.S. hospitals that perform emergency angioplasty.

Previously published guidelines developed by the ACC and the AHA recommend that hospitals treating STEMI patients with percutaneous coronary intervention (PCI) should reliably achieve a door-to-balloon time of 90 minutes or less. However, accomplishing this level of performance is an organizational challenge and many patients are not treated within the guideline recommendation. Thus, improving timeliness of treatment represents an important opportunity to improve the quality of patient care.

Harlan M. Krumholz, MD, SM, FACC, chair of the D2B Working Group and a multidisciplinary team conducted a study funded by the National Heart, Lung and Blood Institute in which 365 hospitals were surveyed to identify strategies that were significantly associated with a faster door-to-balloon time. Data from this study were simultaneously presented at AHA’s Annual Scientific Sessions in Chicago and published in the New England Journal of Medicine.

Several evidence-based strategies have been identified to assist hospitals in reducing their door-to-balloon times. The strategies involve multiple departments and require a systems approach. They include:

Over the coming months, participating hospitals will receive an implementation manual and tool kit, as well as information on how to best construct the team needed to implement the changes; what the roles and responsibilities of each member should be; and how to identify the point person for the project within each primary PCI hospital.

An update on the campaign will be given at the ACC’s Annual Meeting, ACC.07, in New Orleans from March 24-27, 2007.

Most U.S. Hospitals too Slow in Handling Heart Attacks

Most U.S. hospitals do not move fast enough to treat heart attack victims with life-saving emergency angioplasties, according to a study. The study, published in the November 16 New England Journal of Medicine showed that many hospitals did not follow federal guidelines to carry out the procedure in a timely fashion.

The faster doctors carry out the procedure, the higher the probability of survival, according to the study, which coincides with a meeting of the American Heart Association in Chicago.

Researchers from Yale School of Medicine identified several ways to speed up door-to-balloon time. The study, which included 365 U.S. hospitals, showed that the delay in performing angioplasty varied from between 55 and 120 minutes after arriving at the emergency room.

Federal recommendations are for less than 90 minutes. The risk of death rises 42 percent if the procedure is delayed at least 30 minutes, according to the researchers. Slightly more than one-third of heart attack victims in the United States obtain an angioplasty within the first 90 minutes. To gain time, the researchers urge activating the catheterization laboratory while the victim is on his way to the hospital, as well as improving coordination of hospital personnel and having a cardiologist on round-the-clock duty.

Despite the effectiveness of these strategies, a minority of the hospitals surveyed were using them, even though many of the strategies were feasible and could be immediately implemented, said lead author Elizabeth Bradley.

The publication of the research also coincides with the launch of a new campaign by the American College of Cardiology and the American Heart Association to speed up angioplasties and encourage hospitals to implement the strategies. One-third of the approximately 865,000 annual heart attacks in the United States can be treated with angioplasty.

Late Reperfusion Fails to Reduce Cardiovascular Complications

Balloon angioplasty plus stenting failed to reduce major cardiovascular complications in patients who had the procedure three to 28 days after a heart attack, researchers reported at the American Heart Association’s Scientific Sessions 2006. Results of the Occluded Artery Trial (OAT), a National Heart, Lung, and Blood Institute (NHLBI) funded international, randomized clinical study, were presented in a late-breaking clinical trials session. The trial included 2,166 people and is the largest and longest study to compare very late reperfusion to medication alone.

OAT was designed to help resolve a controversy about treatment of over one hundred thousand heart attack survivors in the United States alone each year: whether late opening of blocked coronary arteries would provide any long-term benefits to stable patients, said Judith S. Hochman, MD, OAT study chair and Harold Snyder Family Professor of Cardiology, clinical chief and director of the Cardiovascular Clinical Research Center at the New York University School of Medicine in New York City.

Treatment to reopen totally blocked arteries reduces damage and improves survival if given during the first 12 hours of a heart attack, and up to 36 hours in selected unstable patients. However, an estimated one-third of eligible patients do not receive reperfusion therapy in that timeframe because many of them arrive at the hospital after the treatment window has closed.

In the United States, many physicians favor mechanical opening of totally occluded arteries in stable patients even after the treatment window has closed despite the lack of evidence based on large, randomized, controlled trials on the issue, said Gervasio A. Lamas, MD, study co-chair and director of Cardiovascular Research and Academic Affairs at Mount Sinai Medical Center in Miami, Fla.

OAT tested the hypothesis that percutaneous coronary intervention (PCI) with stenting would be better than medication alone if performed three to 28 days after a heart attack in stable patients who were at increased risk of long-term events death, repeat heart attack and heart failure and who had a totally occluded coronary artery causing the heart attack. Most of these patients had only one coronary artery with severe disease. The primary endpoint was a composite of death, repeat heart attack or heart failure after an average of three years of follow up.

Stenting was required when feasible, with investigators free to choose bare-metal or drug-eluting stents. Most patients did not receive drug-eluting stents. The researchers followed the patients for an average of three years by telephone or through clinic visits and used medical records to check on the clinical events of interest. The endpoints were verified by an independent classification central committee. Maryland Medical Research Institute was the data coordinating center, led by Genell L. Knatterud, PhD.

The study results show no statistically significant differences between the late PCI and medication group in the occurrence of death, heart attacks or heart failure. However, the researchers found an unsettling trend toward more heart attacks in the late PCI group than in the medication group. PCI-assigned patients tended to have excess rates of repeat heart attack, including non-procedure-related events. In addition, elevation of cardiac markers was significantly more common in the PCI group soon after randomization to that treatment arm.

Although the rate of repeat heart attacks did not reach the level of statistical significance, the trend is worrisome, Hochman said. Further analysis and longer follow-up is required to understand the clinical consequences of these heart attacks. These unexpected results are remarkably consistent among all subgroups, including those at highest risk of adverse left ventricular remodeling, such as those with low ejection fraction.

There were fewer patients with angina in the PCI-assigned group at four months and at one year. However, the overall occurrence of angina was low and declined in both groups, and at three years there was no significant difference between groups in the proportion of patients with angina.

OAT included a substudy called the Total Occlusion Study of Canada (TOSCA)-2 Trial. It used repeat pictures of the coronary arteries and left ventricle in the hearts of a subgroup of 381 subjects in the PCI and medication groups, which was led by Vladimir Dzavik, MD., University Health Network, Toronto General Hospital, Canada and Christopher E. Buller, MD, Vancouver General Hospital, Canada. This study found that the opened artery stayed open at one year in most patients who had the angioplasty but their heart function was not better than the medication group. A subset of those who had additional measurements showed less adverse enlargement of the heart in the PCI group. However, this did not translate into clinical benefit. There may be competing processes whereby repeat heart attacks offset this potential benefit.

Results of the 2,166-patient OAT Trial found no reduction in major cardiovascular events over an average follow-up of three years and a signal of excess non-fatal heart attack when routine PCI was performed on stable patients who still have an occluded IRA following a heart attack, Hochman said. Our findings should lead to lower rates of unnecessary coronary interventions in this specific group of stable patients, which should result in substantial healthcare cost savings. Early treatment of heart attacks remains a critically important therapy.

Drug-eluting Stents May Restore Blood Flow to Lower Legs

Drug-eluting stents may be a feasible and safe treatment option to restore and maintain blood flow through small occluded vessels in the lower leg, according to a small, one-year study reported at the American Heart Association’s Scientific Sessions 2006.

Initial experience with 10 patients in this study indicates that drug-eluting stents reduce the risk of restenosis. There was no restenosis in 90% of participants. This is in contrast to the expected 50% of patients who have required treatment for restenosis in previous studies, researchers said.

This is a very common problem that is associated with significant morbidity and mortality, said Arthur G. Grant, MD, the study’s lead author and an interventional cardiology fellow at Ochsner Medical Center in New Orleans. It is something that we need to do a better job of treating. This is an exciting first step toward getting some answers to a difficult problem.

In this study, Grant reported findings from the first 10 patients, all of whom have been followed for at least a year. One patient had claudication and nine had severe arterial narrowing that placed them at risk of amputation. Nine patients didn't require additional procedures to open the artery after stent placement and haven’t had complications for at least a year. The one treatment failure involved a patient who developed a blood clot in the stent three weeks after it was placed and required emergency angioplasty to restore blood flow.

Obviously, we need to study more patients, Grant said. We have placed drug-eluting stents in several other patients, but we haven’t followed them long enough to determine the outcome. Even though only a few patients have been treated, the results are really encouraging.

Heart Valves Grown From Womb Fluid Cells

Scientists for the first time have grown human heart valves using stem cells from the fluid that cushions babies in the womb. The idea is to create these new valves in the lab while the pregnancy progresses and have them ready to implant in a baby with heart defects after it is born. The Swiss experiment follows recent successes at growing bladders and blood vessels and suggests that people may one day be able to grow their own replacement heart parts in some cases, even before they’re even born.

It’s one of several tissue engineering advances that could lead to homegrown heart valves for infants and adults that are more durable and effective than artificial or cadaver valves.

Heart valve defects can be detected during pregnancy with ultrasound tests at about 20 weeks of pregnancy. At least one-third of afflicted infants have problems that could be treated with replacement valves, said Dr. Simon Hoerstrup, a University of Zurich scientist who led the work, which was presented at the American Heart Association conference.

Conventional procedures to fix faulty heart valves all have drawbacks. Artificial valves are prone to blood clots and patients must take anti-clotting drugs for life. Valves from human cadavers or animals can deteriorate, requiring repeated open-heart surgeries to replace them, Hijazi said. That's especially true in children, because these valves don't grow along with the body. Valves made from the patient's own cells are living tissue and might be able to grow with the patient.

The Swiss procedure has another advantage: using cells the fetus sheds in amniotic fluid avoids controversy because it doesn't involve destroying embryos to get stem cells.

Amniotic fluid was obtained through a needle inserted into the womb during amniocentesis. Fetal stem cells were isolated from the fluid, cultured in a lab dish, then placed on a mold shaped like a small ink pen and made of biodegradable plastic. It took only four to six weeks to grow each of the 12 valves created in the experiment. The researchers said lab tests showed they appeared to function normally.

The next step is to see if they work in sheep, a two-year experiment that Hoerstrup said is under way. Hoerstrup said amniotic stem cells also can be frozen for years and could potentially be used to create replacement parts for aging or diseased valves in adults, too.

The research is preliminary and experts say implanting tissue-engineered human valves in human hearts is likely years away. Dr. John E. Mayer Jr., a Children’s Hospital Boston heart surgeon and tissue engineering pioneer, said the big question is whether stem cells from amniotic fluid can create valves superior to those made from other cell types.

Working with dogs and using a 64-slice CT scanner, Johns Hopkins heart specialists have developed a means of tracking blood that has been slowed down by narrowing of the coronary arteries. Researchers say it took them less than half the time of exercise stress tests and echocardiograms.

The Hopkins team presented their initial findings at the American Heart Association’s annual Scientific Sessions. Already, the Hopkins technique, in which patients are given a drug to stress their heart during the scan, is undergoing clinical testing. Results among 60 patients are expected within a year.

If the human trials prove equally successful, senior investigator Albert C. Lardo, PhD, an assistant professor at The Johns Hopkins University of School of Medicine and its Heart Institute, says that CT scanning could dramatically change the way we diagnose coronary disease in patients with initial symptoms of chest pain, by providing a safe, non-invasive and fast method to detect blood-flow problems in heart tissue.

Because it takes less than 15 minutes to perform and does not require patients to be stabilized ahead of scanning, it could replace most other more time-consuming tests that help find blockages, including not only exercise stress testing and echocardiograms, but also positron electron tomography (PET) imaging or magnetic resonance imaging, he says.

The new technique could also help eliminate many unnecessary invasive catheterization procedures when there is no underlying blockage, or become a practical test to verify if treatments with drugs therapies, surgical bypass or stented arteries have worked to improve blood flow.

Lardo, a biomedical engineer who specializes in perfusion imaging, says the tiny size of blood vessels, many no bigger than 0.5 millimeters in diameter, often makes it difficult for physicians to pick up the subtle clues that signal the presence of arterial disease. He notes that blood vessels begin to narrow long before mild chest pain and other symptoms occur, leading to reduced blood flow that gradually starves the heart of needed oxygen and nutrients.

Even when patients have a normal exercise stress test, they may still be in the early stages of atherosclerosis, when vessels start to clog, narrow and harden, gradually straining circulation, says lead study investigator Richard George, MD, a Reynolds Foundation postdoctoral cardiology research fellow at Hopkins. The main drawback to the Hopkins test, George says, is that CT imaging, like PET, also exposes patients to radiation from X-rays, but PET takes nearly one hour to perform, whereas CT scanning is quicker.

Researchers compared 64-slice CT scanning with an existing test that is considered to be the gold standard and relies on muscle absorption of microscopic beads in the heart muscle. Six dogs had surgery to place a clamp around a main artery, cutting in half the blood flow to the heart. After surgery, the dogs were injected with the drug adenosine to speed up their heartbeat and maximize circulation. The dose, they note, was the same as that typically used in humans for routine stress testing: 140 micrograms per kilogram per minute, for five minutes.

When forced to pump faster, healthy heart tissue and surrounding blood vessels will adapt, while unhealthy, blocked ones will slow blood flow. These changes are not usually perceptible to the human eye from the scanned images, but can be detected and quantified with CT imaging and computer analysis. Adenosine is commonly used in stress testing, Lardo says, to relax blood vessels in the muscle, which enhances the contrast between normal and abnormal regions of the heart.

After being injected, each of the dogs had a 64-CT scan of its heart and then underwent the standard test for blood flow with chemical beads. The beads, no bigger than 20 micrometers in diameter and called microspheres, were injected into the animal’s bloodstream. Previous research has shown that they will lodge into the heart muscle at a fixed rate compared to how fast blood is flowing. Researchers can actually count the number of beads absorbed into the heart tissue to calculate blood flow.

However, calculating blood flow from the 3-D, scanned CT images was more complicated. Researchers used deconvolution mathematical formulae to gauge the speed of blood flow through the heart and its feeding arteries, and made separate calculations for each of the organ’s three major regions: the front, back and side walls.

When researchers compared the deconvolution measurements with those derived from the microspheres test, they found the two to be almost identical, with statistical R-values of 0.93 to 0.96, with 1.0 meaning a perfect match.

George conducted the study from June to September 2006 and adds that larger clinical studies are planned at Hopkins to further refine the use of the latest CT technology for diagnosis of heart disease and other ailments. The 64-CT scan has only been available in North America since February 2005. Each machine costs between $1.5 million and $2 million. A single test costs approximately $700. The study was supported by the Donald W. Reynolds Foundation; Toshiba, the manufacture of the 64-CT scanner used in the study; and Astellas Pharma Inc., which makes adenosine, the chemical used in stress testing.

Unique Anticoagulant-antidote Combo Proves Safe in First Human Use

A novel anti-clotting drug and an antidote designed to quickly halt its anticoagulation effects were safe in their first use in humans, researchers reported in a special symposium at the American Heart Association’s Scientific Sessions 2006. The targeted drug-antidote system, known as REG1, holds the promise of a safer and more effective way to reduce the risk of blood clots that can form during many medical procedures, including bypass heart surgery, angioplasty and kidney dialysis.

There were no major safety concerns attributed to the drug, the antidote or their combination, said Richard C. Becker, MD, co-author of the study and a professor of medicine and director of the Cardiovascular Thrombosis Center at Duke University Medical Center in Durham, NC.

The drug-antidote approach was studied in 85 healthy people (average age 32, 34% women). The successful results have already led to a new trial in patients with stable coronary heart disease.

Several anticoagulant agents are in use, including heparin. However, it is difficult to judge heparin’s correct dose and, therefore, its intensity of anticoagulation. Moreover, in 1 to 3% of patients, heparin induces a decrease in platelets, which results in blood clots that can cause a heart attack, stroke or pulmonary embolism. The drug used as an antidote to heparin can trigger cardiovascular side effects and allergic reactions even fatal reactions on rare occasions.

With many existing limitations of traditional anticoagulants, we recognized an opportunity to develop a new system a new paradigm for safe, patient-specific and potentially disease-specific care. We believe that is what we’ve done, Becker said.

The development of REG1 is at the cutting edge of molecular research aimed at creating drugs that work only on one specific molecule. The anticoagulant, a protein-binding, single-stranded nucleic acid called an aptamer, targets activated factor IXa, one of several key molecules involved in the regulation of blood clotting.

The word aptamer is derived from Latin and means ‘to fit,’ Becker said.

The REG1 aptamer folds into a three-dimensional shape, fitting precisely into one target factor IXa. When it locks into that molecule, the drug blocks biologic activity and participation in a sequence of coordinated steps culminating in thrombin generation.

The drug’s antidote is a complementary nucleic acid, which binds rapidly and actively to the aptamer, changing shape and no longer inhibiting factor IXa activity. The ability to reverse the clotting is another novel function of the drug-antidote system.

The REG1 technology was developed at Duke University, where researchers have also developed aptamers targeted to clotting factors VIIa and Xa, and a variety of functionally important proteins on platelets.

Researchers recruited and carefully screened the healthy volunteers who entered the multiphase, randomized study. Participants were first assigned to receive a single injection of the antidote or a placebo. We wanted to first make sure the antidote was safe, Becker said. Then patients were randomized to receive 15, 30, 60 or 90 milligrams of the drug, followed three hours later by the antidote. In the final phase of the study, they were given the drug or placebo.

Among their key findings, the scientists reported:

There was no difference in bleeding between those who received the anticoagulant, the antidote, the combination, or the placebo.

None of the volunteers experienced side effects associated with activation of the complement system.

No antibodies to the genetic material used in the REG1 system were found, indicating the anticoagulant and antidote pose no risk of causing an autoimmune disease, one in which the immune system attacks the body’s own cells.

The anticoagulant showed a definite dose-response. At three dose levels (30, 60 and 90 milligrams), it produced a measurable anticoagulant effect and the higher the dose, the greater the effect.

The antidote worked as designed. Researchers found a prompt and durable reversal of the anti-clotting effect induced by the drug within one to five minutes after injection.

From the new REG1 trial now enrolling patients at five academic institutions, researchers hope to gain greater insight into the system’s safety for patients with coronary artery disease. The study will examine the drug-antidote combination in 50 patients with stable heart disease who are age 55 or older and who are taking aspirin and/or the drug clopidogrel to prevent clotting.

The study was funded by Regado Biosciences Inc., the corporation developing the technology.

Blood Transfusions Should be Used in Moderation for Acute Coronary Syndrome

In a study of more than 44,000 patients being treated for a possible myocardial infarction, cardiologists at the Duke Clinical Research Institute found that while transfusions were associated with a benefit in some patients, they were associated with harm in others.

This finding of harm with transfusions in general is not new, but extends the suggestive evidence from patients in clinical trials to "real-life" patients seen in the community, the researchers said.

The findings further suggest that providers should reconsider their decision-making process about which patients with acute coronary syndrome (ACS) should get transfusions, said the study’s lead investigator, Karen Alexander, MD. She presented the study results at the annual scientific sessions of the American Heart Association.

The researchers looked at hematocrit to see when physicians made the decision to transfuse blood, and they then compared this transfusion decision point to the health outcomes of the patients. For males, the normal hematocrit range is 42 percent to 52 percent, and for women the normal range is 36 percent to 48 percent.

Alexander and colleagues examined how hospitals nationwide treated patients with ACS with transfusions based on their lowest recorded hematocrit. These patients either were anemic when they arrived at the hospital or lost a significant amount of blood while being treated. In both cases, physicians typically give the patients blood transfusions.

The researchers found that while transfusions were beneficial in those whose nadir hematocrit, or lowest level, was less than 24 percent, transfusions were associated with greater harm in those whose nadir hematocrit was greater than 30 percent, Alexander said. For patients with a hematocrit between 24 percent and 30 percent, the researchers found that transfusions were associated with no benefit or no harm.

Our data suggests that providers may want to reconsider how they decide which patients should get transfusions, Alexander said. Our data confirms no harm or benefit in the medium range of 24 percent to 30 percent, so in this group of patients, it might be best to wait and see if the hematocrit drops farther before making the decision to transfuse. Given the scarcity of the blood supply, we certainly want to apply this therapy in those who stand to benefit the most while at the same time avoiding harm.

For the analysis, Alexander and colleagues drew on a national database called CRUSADE, which is coordinated by the Duke Clinical Research Institute and contains patient information from more than 400 hospitals. In total, the researchers identified 44,242 patients treated for acute coronary syndrome from 2004 to 2005. Of this population, 10.4 percent had received a blood transfusion, and 3.9 percent of the patients died, Alexander said.

In transfused patients with a nadir hematocrit of less than 24 percent, the mortality rate was 12 percent, compared with 15 percent for those who did not receive a transfusion, Alexander said. The mortality rates for patients with a hematocrit of 24 percent to 30 percent were similar whether they were transfused or not.

The reasons why transfusions may cause harm are unclear, Alexander said. The red blood cells may be depleted of nitric oxide, which helps deliver oxygen from the cells to tissues but which degrades quickly in stored blood. It also is possible that transfused blood may stimulate an immune response that exacerbates already existing heart disease. A randomized study is needed to clarify the safety and benefit of transfusion, just as other therapeutic interventions for ACS are tested, Alexander said.

Major Advance in Tissue Engineering Growing Heart Valves

For the first time, researchers have successfully used a rabbit’s cells to grow heart-valve-shaped tissue inside the animal’s body, according to research reported at the American Heart Association’s Scientific Sessions 2006. The process may someday make it possible to grow rejection-proof replacement valves for humans using a person’s own cells, a process called autologous tissue engineering.

It is the first fabrication of an autologous heart valve inside a living body, said Kyoko Hayashida, MD, lead author of the study and a research fellow at the National Cardiovascular Center Research Institute in Osaka, Japan, and at the Kyoto Prefecture University of Medicine.

We created an autologous valved-conduit through a simplified and less costly process carried out in living bodies, she said. If every body organ could be recreated by using autologous cells, it would solve the current shortage of donated organs available for transplantation and the use of costly and harmful anti-rejection drugs.

In body tissue-engineering takes advantage of the body’s tissue encapsulation phenomenon, in which the body’s cells naturally surround implanted foreign materials, she said.

Damaged or deformed heart valves are replaced with donated valves, mechanical heart valves or valves from other species such as pigs (xenografts).

All of those approaches have drawbacks, researchers said. Mechanical valves are prone to blood clots. As a result, patients with mechanical valves must take anti-clotting drugs for the rest of their lives. Valves from human or animal donors may gradually deteriorate because they have no living cell components and they can’t self-repair. Furthermore, none of those replacement heart valves can grow as the recipient’s body does, making them problematic for use in children born with faulty heart valves, Hayashida said.

Tissues made from the patient’s own cells hold the promise of growing along with the patient, she said. Hayashida said that her research group developed plastic molds that included three flap-like leaflets that mimic the flaps of a heart valve. The leaflets contained a tiny opening, less than one millimeter wide. An elastic-like conduit scaffold, repeatedly pierced by a laser to give it a sponge-like texture, held the leaflets in place.

The entire apparatus is just over a centimeter long with a diameter of less than a centimeter, making it possible to implant up to five molds in a layer of fat on the rabbits’ backs without bothering the rabbits, who went about their usual activities, she said.

The laser-produced holes allowed the rabbits’ cells to infiltrate the molds and grow all around them, she said. After one month, the researchers removed the molds from the rabbits, again without incident to the animals. Hayashida and her colleagues then removed the outer mold, and left intact the heart-valve-shaped inner mold, now surrounded by tissue and attached by more new tissue to the donut-shaped conduit of tissue surrounding it.

They implanted 10 molds: five in the first rabbit, three in the second rabbit and two in the third. They reported a 50 percent success rate overall (in five of 10 attempts). Although the valve conduits did not have the same cell layers as natural heart valves, they functioned in a similar way when researchers performed flow studies in test tubes.

Future research will investigate whether the valves can resist the fluid pressures encountered by native heart valves without degradation. The researchers also plan to further evaluate the engineered valve’s function when implanted in the body, as well as its potential for growth, self-repair and regeneration in the body, she said.

In a poster session, one of Hayashida’s colleagues, Taiji Watanabe, MD, reported that the same autologous tissue engineering approach was used to create small-caliber (less than 2 millimeter diameter) blood vessel grafts, called Biotubes. Biotubes performed like native arteries when implanted in the rabbits in which they were grown. The biotubes also withstood high pressures and showed no signs of rupture during three months of follow-up after implantation, Watanabe said.